Approximately 25% of all human cancers result from a mutant gene that encodes a mutant form of the protein known as Ras. In cancer cells, Ras activates the cells to divide in an unrestrained manner. To induce cell division, Ras must be localized at the inner surface of the cancer cell membrane. This membrane localization of Ras is effected by attachment of a hydrophobic group, typically the farnesyl group, but possibly the related geranylgeranyl group. In either case, the group becomes attached to Ras enzymatically in a process known as prenylation. Thus, interference with prenylation of Ras has the potential to prevent Ras localization at the inner surface of the cancer cell membrane, resulting in the cessation of unrestrained cell division and/or reversion of the cancer cell to a normal phenotype.
The enzyme that attaches the farnesyl group to Ras, RhoB, and other proteins to facilitate the proper localization of these proteins in the cell is farnesyl protein transferase, also known as protein farnesyltransferase (referred to here as FTase). The farnesyl group becomes attached to Ras, RhoB, and other proteins by reaction with farnesyl diphosphate, also known as farnesyl pyrophosphate (referred to here as FPP). In other words, FTase catalyzes the reaction illustrated below for the Ras protein, in which the protein becomes attached to the farnesyl group by displacement of pyrophosphate (P2O74−, referred to here as PPi):FTaseRas+FPP→farnesyl-Ras+PPi
Thus, a key target in a strategy to retard cancer cell proliferation is the enzyme FTase. By regulating FTase activity, Ras farnesylation, RhoB farnesylation and geranylgeranylation, and the prenylation of other proteins can be controlled. This can alter the intracellular distribution of these proteins and in turn prevent cancer cells from proliferating. Because normal cells also require FTase activity, the optimal regulation of prenyltransferase activity must be determined empirically.
Many substances are known to block FTase activity and prevent farnesylation of cellular proteins. These include inhibitors of the enzyme FTase, which generally operate by blocking the binding of proteins to be prenylated, FPP, or both, to the FTase active site. Without the ability of the normal substrates (e.g. Ras and FPP) to bind to FTase, this enzyme can no longer transfer the farnesyl group from FPP to Ras. In general, inhibitors structurally mimic one or both of the natural substrates of the enzyme, in this case Ras and/or FPP. For conventional inhibitors, their binding to FTase is reversible and noncovalent (i.e. the binding of the inhibitor to FTase does not involve the formation of covalent bonds). Instead, hydrophobic forces, hydrogen bonding, electrostatic attraction, etc. are principally responsible for binding of the inhibitor to the enzyme FTase. These binding forces allow the inhibitor to block the site on FTase where the normal substrates need to bind for farnesylation of Ras to occur.
It would therefore be desirable to develop a method of preventing, substantially irreversibly, FTase from farnesylating Ras and, more generally, preventing other prenylation enzymes from promoting the inner cell membrane localization of oncoproteins. Interaction of FTase, for example, with substances that covalently modify the active site of FTase should result in an enzyme with an essentially permanent reduction in catalytic ability. In principle, and in contrast to conventional enzyme deactivation, the covalent attachment can be irreversible or nearly irreversible. The desirable characteristics of a prenylation enzyme inhibitor may include both a substrate-mimicking group as well as a group having the ability to bond covalently to the enzyme at or near its active site.